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BMP Signaling Inhibition in Drosophila Secondary Cells Remodels the Seminal Proteome and Self and Rival Ejaculate Functions

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BMP Signaling Inhibition in Drosophila Secondary Cells Remodels the Seminal Proteome and Self and Rival Ejaculate Functions BMP signaling inhibition in Drosophila secondary cells remodels the seminal proteome and self and rival ejaculate functions Ben R. Hopkinsa,1,2, Irem Sepila, Sarah Bonhamb, Thomas Millera, Philip D. Charlesb, Roman Fischerb, Benedikt M. Kesslerb, Clive Wilsonc, and Stuart Wigbya aEdward Grey Institute, Department of Zoology, University of Oxford, OX1 3PS Oxford, United Kingdom; bTarget Discovery Institute (TDI) Mass Spectrometry Laboratory, Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, OX3 7BN Oxford, United Kingdom; and cDepartment of Physiology, Anatomy, and Genetics, University of Oxford, OX1 3QX Oxford, United Kingdom Edited by David L. Denlinger, The Ohio State University, Columbus, OH, and approved October 22, 2019 (received for review August 22, 2019) Seminal fluid proteins (SFPs) exert potent effects on male and sequestering SFPs in different cells or glands, males are afforded female fitness. Rapidly evolving and molecularly diverse, they control over their release and, consequently, afforded spatiotem- derive from multiple male secretory cells and tissues. In Drosophila poral control over their interactions with sperm, the female re- melanogaster, most SFPs are produced in the accessory glands, productive tract, and with other SFPs (16). Additionally, functional which are composed of ∼1,000 fertility-enhancing “main cells” diversification of tissues and cell types may be required to build and ∼40 more functionally cryptic “secondary cells.” Inhibition of specialized parts of the ejaculate, such as mating plugs (17). In bone morphogenetic protein (BMP) signaling in secondary cells either case, activities may be carried out independently between suppresses secretion, leading to a unique uncoupling of normal cell types and tissues or there may be cross-talk between them female postmating responses to the ejaculate: refractoriness stim- that coordinates global seminal fluid composition. It has been ulation is impaired, but offspring production is not. Secondary-cell suggested that such cross-talk may be required to drive the so- secretions might therefore make highly specific contributions to phisticated strategic changes in ejaculate composition observed the seminal proteome and ejaculate function; alternatively, they in relation to sperm competition threat (18). Fundamentally, might regulate more global—but hitherto undiscovered—SFP func- to understand how ejaculates evolve it is essential that we EVOLUTION tions and proteome composition. Here, we present data that sup- understand the drivers of diversity in the elements within the port the latter model. We show that in addition to previously male reproductive system, as well as the functional connectivity reported phenotypes, secondary-cell-specific BMP signaling inhibi- between them. tion compromises sperm storage and increases female sperm use The male reproductive system of Drosophila melanogaster efficiency. It also impacts second male sperm, tending to slow entry consists of testes that produce sperm, and 3 secretory tissues that into storage and delay ejection. First male paternity is enhanced, which suggests a constraint on ejaculate evolution whereby high Significance female refractoriness and sperm competitiveness are mutually ex- clusive. Using quantitative proteomics, we reveal changes to the seminal proteome that surprisingly encompass alterations to How are ejaculates built? Fertility-enhancing seminal fluid is main-cell–derived proteins, indicating important cross-talk between the product of many different glands and cells, but how the classes of SFP-secreting cells. Our results demonstrate that ejaculate function and composition of seminal fluid emerges from these composition and function emerge from the integrated action of different elements is poorly resolved. Here, we characterize the multiple secretory cell types, suggesting that modification to the contributions of the functionally cryptic Drosophila accessory cellular make-up of seminal-fluid-producing tissues is an important gland secondary cells to ejaculate composition and reproduc- factor in ejaculate evolution. tive outcome. We find that in adults these cells are central to the regulation of the seminal proteome, the promotion of normal sperm behavior, and the induction of many, but not all, reproduction | seminal fluid | sexual selection | sperm competition | sperm postmating responses. Our results illustrate interdependency between glandular cell types, identify constraints in ejaculate jaculates are compositionally rich. In addition to sperm, functions linked to male reproductive success, and provide in- Emales transfer a mixture of proteins (seminal fluid proteins sights into the design and production of ejaculates. [SFPs]), lipids, salts, vesicles, and nucleic acids, which together – constitute the seminal fluid (1 3). The phenotypic effects of Author contributions: B.R.H., I.S., and S.W. designed research; B.R.H., I.S., S.B., T.M., and seminal fluid in females are broad, particularly in invertebrates. S.W. performed research; S.B., P.D.C., R.F., B.M.K., and C.W. contributed new reagents/ In various species these effects include increased aggression, analytic tools; B.R.H. analyzed data; and B.R.H. and S.W. wrote the paper with contribu- reduced sexual receptivity, shifts in dietary preference, confor- tions from all authors. mational changes in the reproductive tract, immunomodulation, The authors declare no competing interest. and stimulation of offspring production (reviewed in refs. 4–6). This article is a PNAS Direct Submission. A number of SFPs have been further implicated in sperm com- This open access article is distributed under Creative Commons Attribution License 4.0 petition, the process by which sperm from different males compete (CC BY). for fertilizations (7–10). Consequently, seminal fluid represents a Data deposition: The proteomics data reported in this paper have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository (dataset identifier critical mediator of male reproductive success (11, 12). PXD015253). All other data are deposited in the Oxford University Research Archive While sperm are always produced in testes, seminal fluid (https://ora.ox.ac.uk) with DOI:10.5287/bodleian:nb20wgNw8. generally comprises products drawn from a number of repro- 1To whom correspondence may be addressed. Email: [email protected]. ductive tissues (13). These tissues vary considerably in number, 2Present address: Department of Evolution and Ecology, University of California, Davis, cellular make-up, and developmental identity among species, CA, 95616. with lineages showing evolutionary patterns of loss, modification, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and acquisition (4, 13–15). Why male reproductive systems in- 1073/pnas.1914491116/-/DCSupplemental. corporate this diversity is unclear. It has been suggested that by www.pnas.org/cgi/doi/10.1073/pnas.1914491116 PNAS Latest Articles | 1of10 Downloaded by guest on October 1, 2021 contribute to the seminal fluid: the paired accessory glands, ejac- Results and Discussion ulatory duct, and ejaculatory bulb (4) (Fig. 1A). The majority of Sperm Storage Is Compromised in Dad-Mated Females. We began by the ∼200 SFPs known to be transferred to females are produced mating virgin females to males who possessed GFP-tagged sperm and stored in the accessory glands (19). Each of the 2 lobes of the (33) and who overexpressed the transcriptional repressor of glands is composed of 2 distinct cell types (20). The majority are BMP signaling Dad, which suppresses secondary-cell secretion the ∼1,000 small, binucleate “main cells” (20), which are thought (31) (hereafter “Dad” males), to test whether these secretions to produce most of the gland’s secretion (21). Accordingly, these are required for normal sperm entry into storage. We found no cells have been shown to be the sole production site for several significant difference between Dad and control males in mating highly abundant and functionally important SFPs, including sex duration (linear model [LM], F1, 110 = 0.074, P = 0.787; SI Ap- peptide (SP), a key driver of postmating changes (22–25). Ab- pendix, Fig. S1) or in the number of sperm transferred (LM, lation of main cells leads to failures in the induction of the main F1, 53 = 1.700, P = 0.198; Fig. 2A). The variance in sperm transfer female postmating responses: receptivity to remating remains was high for both genotypes, but was consistent with previous high, and egg production unstimulated (26). reports for D. melanogaster (34) and other Diptera (35). The The distal tips of each gland also contain a further sub- proportion of sperm that initially enters into the storage organs population of ∼40 unusually large “secondary cells” (refs. 20 and (seminal receptacle and paired spermathecae), and that is ulti- 27 and Fig. 1B). As with main cells, failures in normal secondary- mately stored (5-h postmating; ref. 33) was significantly lower in cell development are associated with defective postmating re- Dad-mated females (initial entry at 25 min, generalized linear sponses: high receptivity, low fecundity (28, 29). This is partly model [GLM], F = 5.340, P = 0.024; Fig. 2B; 5-h storage, LM, “ ” 1, 53 attributable to glycosylation defects in SP network proteins, F1, 53 = 5.043, P = 0.029; Fig. 2C). This demonstrates a role for which are required for the storage and gradual release of sperm- secondary-cell activity in promoting
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